Process for the purification of a growth factor protein

ABSTRACT

A process of purifying a Growth Factor Protein in a purification sequence employing chromatography characterized in that
         at least one chromatography is performed using a multimodal resin   the Growth Factor Protein binds to the multimodal resin at a pH between 4 to 6.2, and the Growth Factor Protein is eluting at a pH &gt;6.3, and the elution of Growth Factor Protein is improved by addition of arginine and/or NaCl to the eluting buffer.   The multimodal resin step is followed by a yeast derived affinity ligand resin step, which results of a purity of the product &gt;90%.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/638,244,filed Sep. 28, 2012, published Apr. 18, 2013, as US2013/0096279,currently pending, which is a U.S. National Stage of PCT PatentApplication No. PCT/EP2011/054920, filed Mar. 30, 2011, which claims thebenefit of U.S. Provisional Patent Application No. 61/282,894, filedApr. 16, 2010, and which claims the benefit of European PatentApplication No. 10158522.2, filed Mar. 30, 2010, all of which areincorporated by reference in their entireties herein.

BACKGROUND OF THE INVENTION

The present invention pertains to a process of the purification of agrowth factor protein employing chromatography.

The purification of proteins from sources of natural origin is achallenge as the protein of interest is often only present in traceamounts and accompanied by other biopolymers such as lipids, proteins,or even cell fragments. Moreover the proteins of interest are mostlyassociated with a biological function which is often lost during processsteps for its purification.

The arsenal of methods for purifying biopolymers such as proteins islarge. Besides precipitation methods chromatographic methods on variouskinds of materials are known. Frequently the materials are modified withchemical moieties such as organic ions, cations such as protonatedamines or partially or completely alkylated amines. Such materials areused as anion exchangers. But also cation exchangers can be used forpurification methods depending on the physical properties of the proteinof interest such as shape, molecular weight and in particular itscharge. Alternatively or in combination affinity chromatography isemployed.

It is known in prior art that one disadvantage with traditional ionexchange chromatography resins (as for example SP-, CM-, Q- or DEAESepharose FF ion exchange chromatography resins) is that the binding ofa protein to the resin only can be performed within relatively low saltconcentration (conductivity, osmolality etc.), typically in the range of0.01-0.15M of salt (NaCl etc.) concentration. In certain applicationsthere would be a demand to be able to use the relatively mildpurification conditions a ion exchange chromatography step exertstowards the proteins, also directly (without further dilution) to achromatography resin at somewhat increased ionic strength. An increasedionic strength can be of significant advantage for the protein stabilityin a protein solution; especially in a crude protein preparation like inthe harvest of recombinant produced protein products or in plasmaderived products where potential proteases are present in the solutionwhich can affect the target protein negatively. As proteases often workbest at physiological conditions (like is the case in most cellsystems), i.e approximately pH 7 and a salt concentration ofapproximately 0.15M.

Proteases could be inhibited by changing the work-up conditions eg byaddition of salt and/or change of the pH, however, both these parametersare critical for the performance of a conventional ionic chromatographystep and thus often impossible to use in their combination. There is aneed to provide a purification method in the course of which conditionsto minimise the effects of proteases can be employed.

WO-A2-2008/073620 discloses a manufacturing method for polypeptides thatare produced in insect cells using a baculoviral expression system. Inone example, the insect cell culture is supplemented with a lipidmixture immediately prior to infection (e.g., one hour prior toinfection). The polypeptides are isolated from the insect cell cultureusing a method that employs anion exchange or mixed-mode chromatographyearly in the purification process. This process step is useful to removeinsect-cell derived endoglycanases and proteases and thus reduces theloss of desired polypeptide due to enzymatic degradation. In anotherexample, mixed-mode chromatography is combined with dye-ligand affinitychromatography in a continuous-flow manner to allow for rapid processingof the insect-cell culture liquid and capture of the polypeptide. In yetanother example, a polypeptide is isolated from an insect cell cultureliquid using a process that combines hollow fiber filtration, mixed-modechromatography and dye-ligand affinity in a single unit Operationproducing a polypeptide solution that is essentially free ofendoglycanase and proteolytic activities. In a further example, theisolated polypeptides are glycopeptides having an insect specificglycosylation pattern, which are optionally conjugated to a modifyinggroup, such as a polymer (e.g., PEG) using a glycosyltransferase and amodified nucleotide sugar.

WO-A2-2009/063069 discloses a process for purifying peptides, inparticular but not exclusively, to a process for removing endotoxinsfrom a peptide solution, to a kit comprising reagents for said processand to the purified peptide obtained by said process.

Dasari Venkata Krishna Rao et al. discloses a purification methodemploying a process control-strategy developed for improving the yieldof rhG-CSF (recombinant human granolocyte colony-stimulating factor). Apurity of 99% with an overall yield of 2.18 g/I was achieved in thepresent study. Analysis of the product during purification indicatedthat detergents removed 72% of LPS (lipopolysaccharides) and 98% of HCPs(host cell proteins) without removing nucleic acid. Cysteineconcentration was a key parameter in protein refolding. The bed heightand HETP (height equivalent theoretical plates) value in the SEC(size-exclusion chromatography) column was evaluated and its impact onthe resolution was studied. Formulation during SEC was found to becrucial for increasing the product yields with saving of time andprocess costs.

Quan Bai et al. studies the renaturation and purification of recombinanthuman granulocyte macrophage colony stimulation factor (rhGM-CSF)expressed in Escherichia coli with strong anion-exchange chromatography(SAX). The effects of pH values, ratios of concentrations of GSH/GSSG,and urea concentrations in the mobile phase on the renaturation andpurification of rhGM-CSF with SAX were investigated, respectively. Theresults show that the above three factors have remarkable influences onthe efficiency of renaturation and mass recovery of rhGM-CSF. Theaddition of GSH/GSSG in the mobile phase can improve the formation of ofcorrect disulfide bonds in rhGM-CSF so that its renaturation yieldincreases. In addition, to enhance the mass recovery of rhGM-CSF withSAX, the low concentration of urea was added in the mobile phase toprevent denatured protein aggregation. Under the optimal conditions,rhGM-CSF was renatured with simultaneous purification on SAX columnwithin 30 min only by one step.

Shelly A. Pizarro reports about a vascular endothelial growth factor(VEGF165) which is a potent mitogen that induces angiogenesis andvascular permeability in vivo and has demonstrated potential intherapeutic applications for accelerating wound healing. The processdescribed in this report involves a bacterial expression system capableof producing approximately 9 g of rh VEGF per liter of broth and adownstream purification process of protein refolding and threechromatography steps prior to formulation of the drug substance. A highcell density (HCD) fed-batch fermentation process was used to producerhVEGF in perisplasmic inclusion bodies. The inclusion bodies areharvested from the cell lysate and subjected to a single-step proteinsolubilization and refolding operation to extract the rhVEGF forpurification. Overall recovery yields observed during development,including refolding and chromatography, were 30±6%. Host cell impuritiesare consistently cleared below target levels at both laboratory andlarge-scale demonstrating process robustness. The structure of therefolded and purified rhVEGF was confirmed by mass spectrometry.N-terminal sequencing, and tryptic peptide mapping while productvariants were analyzed by multiple HPLC assays.

Kimberly A. Kaleas discloses that mixed-mode chromatography resins aregaining popularity as purification tools for challenging feedstocks, anddiscloses the development of an industrial application to selectivelycapture recombinant human vascular endothelial growth factor (rhVEGF) onCapto MMC from an alkaline feedstock. Capto MMC resin contains a ligandthat has the potential to participate in ionic, hydrophobic, andhydrogen bonding interactions with proteins and is coupled to a highlycross-linked agarose bead matrix. VEGF is a key growth factor involvedin angiogenesis and has therapeutic applications for wound healing. Itis expressed in Escherichia coli as inclusion bodies. Solids areharvested from the cell lysate, and the rhVEGF is solubilized andrefolded and pH 9.8 in the presence of urea and redox agents. The uniquemixed mode characteristics of Capto MMC enabled capture of this basicprotein with minimal load conditioning and delivered a concentrated poolfor downstream processing with >95% yields while reducing host cellprotein content to <1.2%. This study explores the impact of loadingconditions and residence time on the dynamic binding capacity as well asthe development of elution conditions for optimal purificationperformance. After evaluating various elution buffers, L-arginine HClwas shown to be an effective eluting agent for rhVEGF desorption fromthe Capto MMC mixed-mode resin since it successfully disrupted themultiple interactions between the resin and rhVEGF. The lab scale effortproduced a robust chromatography step that was successfully implementedat commercial manufacturing scale.

One object of the invention was to avoid the drawbacks of thepurification processes of a growth factor protein of prior art byproviding a novel process.

According to the invention the object is accomplished by a process ofpurifying a growth factor protein selected from the group consisting ofColony Stimulating Factor (CSF) such as G-CSF (Granulocyte ColonyStimulating Factor) or granulocyte-macrophage CSF (GM-CSF), interleukin3 (IL-3), Hepatocyte growth factor, Epidermal growth factor andfibroblast growth factor (acid) in a purification sequence employingchromatography wherein

-   -   at least one chromatography is performed using a multimodal        resin    -   the Growth Factor Protein binds to the multimodal resin at a pH        between 4 to 6.2, and    -   the Growth Factor Protein is eluting from the multimodal resin        at a pH >6.3.

The invention provides a process in which advantageously the effects ofproteases can be minimised. Making it possible to add salt and/or changethe pH in crude protein sample with potential proteases present whichcould degrade the target protein and to process the protein solutionwithout any further measures and bind the target protein to a mixed modechromatography resin and thus providing a optimized step forconcentration and purification of the target protein in a crude sample,making it suitable for further purification downstream using a specificaffinity chromatography step directed towards the target protein, withreduced protease and/or DNA content during the downstream processing.This is of specific importance, to avoid degradation of the targetprotein during purification, making the combination of multimodalchromatography as a capture step in a crude protein solution.

In one embodiment, the chromatography on multimodal resins is combinedwith a yeast derived affinity ligand chromatography step. Thechromatographic step employing the yeast derived affinity ligand, isespecially suitable for the purification of the target protein in highyield and an unchanged molecule integrity (degradation etc.).

The Growth Factor Protein is a Colony Stimulating Factor (CSF) such asG-CSF (Granulocyte Colony Stimulating Factor). This is a member of thehemopoietic regulatory glycoproteins which are involved in the growthand differentiation of hemopoietic cells from stem cells. Growth FactorProteins are granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3),Hepatocyte growth factor, Epidermal growth factor and fibroblast growthfactor (acid). The Growth Factor Proteins all show an IP 61. In afurther embodiment of the invention the multimodal resin comprisesmoieties bound to a matrix and the moieties are able to interact withthe Growth Factor Protein in a mixture by ionic interactions and othertypes of interactions such as hydrogen bonding, hydrophobic andthiophillic interactions.

In a further embodiment of the invention the affinity ligand is a yeastderived F_(ab) fragment directed towards the growth factor protein.

In a further embodiment of the invention the multimodal resin step isprocessed to capture the Growth Factor Protein from a crude proteinsolution whereafter processing the resulting multimodal chromatographyresin eluate to the yeast derived affinity ligand chromatography stepand after elution of the Growth Factor protein from said affinitychromatography step, exerting a purity of more than approximately 90% inrelation to proteins and DNA.

In another further embodiment of the invention the multimodal resin stepand the yeast derived affinity ligand chromatography step is combinedwith other chromatography purification step to exert a purity of morethan 99% in the final Growth Factor Protein product.

In still another embodiment of the invention the mixture comprising theGrowth Factor Protein is a solution.

In yet another embodiment of the invention the Growth Factor Protein isa recombinant Growth Factor Protein.

In yet another embodiment of the invention the Growth Factor Protein isin a crude protein solution including potentially proteases which candegrade the product.

In another embodiment the Growth Factor Protein is eluted by a pHchange >pH 6.3.

In a further embodiment of the invention the elution is performed withan elution agent comprising an amino acid having a basic side chainand/or high ionic strength. Alternatively or in combination the elutioncan also be performed by a pH change. The pH change is performed byadjusting the pH of the elution buffer with for example sodium hydroxideor acetic acid to desired pH and thereafter applying the buffer to themultimodal resin and the ionic strength adjustment can be performed byadding salt, in the elution buffer composition before applying to themultimodal resin, for example salts included in the Hofmeister series,for example sodium chloride and potassium chloride.

According to the invention the concentration of the elution agent is inparticular in the range of from about 0.1M to about 2 M

According to another embodiment of the invention the Growth FactorProtein binds to the multimodal resin at about pH 6.0 whereas the GrowthFactor Protein is eluted from the multimodal resin at a pH about 6.5 orhigher in particular at about pH 7.0. In a further embodiment of theinvention a buffering substance is used comprising preferably at leastone of the substances selected from the group consisting of sodiumcitrate, histidine, 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonicacid (HEPES), 2-(N-Morpholino)ethane sulfonic acid (MES), Tris base andsodium acetate in particular in a range of about pH 4 to about pH 8.

In the process of the invention one non-ionic detergent can be presentin any of the buffers used, which non-ionic detergent is in particularselected from the group consisting of Polysorbates (Polysorbate 20, 40,60, 80) and Pluronic F68.

In a further embodiment of the process of the invention the amino acidcan be selected from the group of amino acid having a basic side chainincluding arginine, lysine and histidine; the organic salts can beselected from the group of KCl and NaCl

In another embodiment of the invention a wash step is performed at a pHin the range of about pH 4 to about pH 6, before eluting the GrowthFactor Protein from the multi modal resin, characterised that the washbuffer includes washing agents comprising an amino acid having a basicside chain and/or high ionic strength, the ionic strength adjustment canbe performed by adding salt, in the wash buffer composition beforeapplying to the multimodal resin, for example salts included in theHofmeister series, for example sodium chloride and potassium chlorideAccording to the invention the concentration of the washing agent is inparticular in the range of from about 0.1M to about 2M.

It can be advantageous to apply the washing buffer to the multimodalresin, to wash away contaminants (proteases, DNA etc.) and retain theGrowth Factor Protein, before the Growth Factor Protein is released.

Particularly, the concentration of the amino acid which is positivelycharged at a pH 6-8 is present in an amount of up to 2M in the washbuffer at a pH of <6.3.

Typically, the amount of arginine is in the range of 0.1-1.0M, inparticular 0.5M in the wash buffer.

In the elution buffer with a pH>6.3 the amount of arginine is typicallyin the range of 0.1 to 2M, in particular 0.5M.

In the elution buffer with a pH≧6.3, sodium chloride is included in arange of 0.1-2.0M, in particular in a range from 0.1 to 1M.

In the wash buffer with a pH<6.3, sodium chloride is included in a rangeof 0.1-2.0M, in particular in a range from 0.1 to 1M.

The amount of non-ionic detergent is typically in the range of 0.001 to1%, in particular in the buffers for multimodal chromatography 0.02%.

The multimodal chromatography resin which can be employed according tothe invention may contain at least one of the following moieties:

-   -   a. a positively charged N-Benzyl-N-methyl ethanolamine ligand,    -   b. a negatively charged 2-(benzoylamino) butanoic acid ligand,    -   c. a phenylpropyl ligand,    -   d. a N-hexyl ligand,    -   e. a 4-Mercapto-Ethyl-Pyridine ligand,    -   f. a        3-((3-methyl-5-((tetrahydrofuran-2-ylmethyl)-amino)-phenyl)-amino)-benzoic        acid ligand or combinations thereof.

In particular, a multimodal chromatography resin for use according tothe present invention is selected from the following commerciallyavailable resins HEP Hypercel™; PPA Hypercel™; Capto Adhere™; CaptoMMC™; MEP Hypercel™.

In another embodiment of the present invention the purification sequencemay further comprise pathogen removal/inactivation steps comprising achemically based inactivation step, a size based removal step,chromatography steps or combinations thereof which steps are based ondifferent physiological properties directed to the pathogen to beremoved.

In a particular embodiment the process of the invention the purificationsequence further comprises the following steps:

-   -   1. a cation multimodal resin such as Capto MMC;    -   2. a chemically based inactivation step for enveloped viruses in        particular the solvent/detergent-inactivation employing        tri-n-butyl phosphate and Triton X-100 as disclosed in EP-A-131        740;    -   3. an affinity resin based on a ligand expressed in yeast;    -   4. a cation exchanger such as SP Sepharose or Resource S;    -   5. a pathogen filtration removal step with a mean pore sized of        about 20 nm such as Planova 20N;    -   6. a buffer exchange and/or concentrating step such as ultra        filtration with an approximate cut off of 1-5 kDa;    -   7. a size exclusion chromatography resin such as Superdex 75.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer.

FIG. 2 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer.

FIG. 3 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer.

FIG. 4 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 of the start material using a sodium acetate buffer.

FIG. 5 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium citrate buffer.

FIG. 6 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 5.0 using a sodium citrate buffer.

FIG. 7 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 5.5 using a sodium citrate buffer.

FIG. 8 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 6.0 using a sodium citrate buffer.

FIG. 9 shows a chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 6.5 using a sodium citrate buffer.

FIGS. 10a and 10b shows a silver stained SDS-PAGE and the separation ofproteins in the start and eluate from the Capto MMC.

FIG. 11 shows a chromatogram of the G-CSF purification from a cell freesupernatant.

FIG. 12 shows a Coomassie stained SDS-PAGE showing the eluate after theaffinity chromatography step.

FIG. 13 shows a silver stained SDS-PAGE showing starting material, flowthrough and eluates after the affinity chromatography step.

The invention is further described by the following non-limitingexamples which have been exemplified by G-CSF purification.

EXAMPLES Description of Analytical Methods Determination of G-CSFContent by G-CSF Specific ELISA

The principle of an Enzyme Linked Immunoabsorbent Assay (ELISA) is thequantification of a protein (antigen) by its specific binding toantibodies against this protein. The G-CSF quantification was performedby use of a sandwich ELISA, based on the G-CSF Duo Set ELISA (R&DSystems, Cat No DY214). As calibration standard E. coli derivedrecombinant human G-CSF (R&D systems, Cat No. 214-CS-005, 0.015-1 ng/ml)was applied. The capturing antibody (mouse anti-human G-CSF) was boundto the wells of a 96-well microtiter plate. After capturing of the G-CSFantigen and a wash step, biotinylated detection antibody (goatanti-human G-CSF) was bound to the G-CSF antigen. After a second washstep, streptavidin conjugated to Horse Raddish Peroxidase(Streptavidin-HRP) was applied, which bounds to the biotinylateddetection antibody. For quantification the peroxidase substrateTetramethylbenzidine (TMB) in presence of hydrogen peroxide was addedand a blue colour develops. After stopping the reaction with sulphuricacid, a stable yellow dye develops. The concentration of yellow dye isproportional to the amount of bound peroxidise and thus proportional tothe amount of G-CSF antigen. The concentration of the dye is measuredphotometrically at 450 nm. The G-CSF concentration in the unknownsamples was calculated from the recombinant human G-CSF standard curve,which always gave a linear correlation coefficient (r) of >0.99.

Determination of G-CSF Content by Reversed-Phase (RP) HPLC

RP-HPLC involves the separation of proteins based on their polarity; theretention of the protein molecules is governed by hydrophobicinteractions between the nonpolar moiety of the solute molecules and thenon polar stationary phase of the HPLC column. An HPLC system (DionexUltimate 3000), equipped with a UV detector and a Jupiter C18, 300 A, 5μm, 4.6×150 mm column (Phenomenex, Cat. no. 00F-4053-EO) was used forprotein determination. The column, run at 20±5 PC, was equilibrated with0.1% (v/v) trifluoroacetic acid (TFA) in water (mobile phase A). Forelution, 0.1% (v/v) TFA in acetonitrile (mobile phase B) was used in thelinear gradient (0-5 min 5% B, 5-12 min 55% B, 12-17 min 100% B, 17-22min 5% B) with a flow rate of 1.0 ml/min. The sample load was 30 μg perinjection in a total injection volume of maximal 100 μL. Detection wascarried out by measuring the UV absorbance at 214 nm. Filgrastim CRSfrom Ph.Eur 2.5-40 μg (2.5-5-10-20-40 μg) was used for the standardcurve. The Filgrastim CRS standard was pre-diluted in water forlaboratory use (WFL) to a concentration of 0.4 mg/ml. A pre-dilution ofthe samples in WFL was performed to a injection amount of 30 μg, ifnecessary. The G-CSF content in the unknown samples was calculated fromthe Filgrastim CRS standard curve, which always gave a linearcorrelation coefficient (r) of >0.99.

Purity Determination by Reversed-Phase (RP) HPLC

The method and devices used for purity determination by RP-HPLC isequivalent to the method for determination of G-CSF content. The purityof the G-CSF containing solution [%] was calculated by setting the peakarea of the G-CSF peak in ratio to the total peak area.

Purity Determination & Molecular Weight Distribution by SDS-PAGE

SDS polyacrylamide gel electrophoresis (SDS-PAGE) involves theseparation of proteins based on their size. The purity determination andanalysis of molecular weight distribution for G-CSF containing sampleswas performed under reduced conditions. For this purpose Tris-Tricinegradient gels (10-20%, from Anamed, Cat No. TR12012) and Tris-HClgradient gels (10-20%, from Biorad, Cat No. 345-0043) were used. For theTris-Tricine gradient gels the Polypeptide SDS-PAGE Molecular WeightStandard from BioRad (Cat No. 161-0326; 1.4-26.6 kDa) was applied asmolecular weight standard; for the Tris-HCl gradient gels the PrecisionPlus Protein All Blue Standard from Biorad (Cat No. 161-0373, 10-250kDa) was applied. The protein bands separated by electrophoresis arevisualized by silver or comassie staining. E. coli derived recombinanthuman G-CSF (non glycosylated, R&D systems, Cat No. 214-CS-005) the theglycosylated, CHO derived commercial product Granocyte (Chugai) wereused as G-CSF references (control samples). The evaluation of molecularweight and purity is done visually by judging the appearances of thestandards, reference (control sample) and analysed samples.

Recombinant G-CSF

Production of G-CSF Containing Cell Suspension and Purification.

Cells

The cell line used is a derivative of human embryonic kidney cell 293(HEK 293), which was adapted to serum-free growth. This host, HEK 293F,was stably transfected with an expression cassette carrying the cDNAcoding sequence for G-CSF. The strong promoter was used for thecassette. The general process is also described in EP 1739179 (Schröderet al).

Cultivation Method

The cells were cultivated in serum-free medium in general equipment andaccording to general methods well known in the art, for example shakenor stirred cultures in t-flasks, shaker flasks and bioreactors(disposable systems and conventional stirred tanks) run as batch,fed-batch, perfusion or continuous chemostat cultures (Freshney, R I(2000), Culture of animal cells: a manual of basic technique, 4^(th) ed,Wiley-Liss; Spier, R E ed (2000), Encyclopedia of cell technology,Wiley, New York; Enfors, S-O and Häggström, L (2000), Bioprocesstechnology: fundamentals and applications, Högskoletryckeriet, RoyalInstitute of Technology, Stockholm; Vinci, V A and Parekh, S R (2003),Handbook of industrial cell culture: mammalian, microbial, and plantcells, Humana Press, USA). Typically, perfusion of medium was used toincrease cell numbers and product titers beyond standard batch culturelevels. The product yield and the amount of host cell proteins differdepending on the cultivation mode:

-   -   the product titre will typically increase with cell numbers    -   the total protein content and DNA content will typical increase        with cell numbers    -   the total protein content and DNA content can also increase with        culture longetivity    -   batch cultures accumulate protein and DNA; nothing is externally        added, nothing is removed    -   perfusion processes rinse cell cultures from metabolites,        protein, DNA and other impurities; filters or cell centrifuges        were typically used for cell retention.

The recombinant product is released from the cells and the cellsuspension or the cell suspension supernatant is the harvest. Theproperties of the harvest (product titres and impurities as mentionedabove) differ depending on the cultivation mode used.

The cell suspension has been used in some of the below described G-CSFexamples.

Purification Method

The recombinant product is released from the cells and the cellsuspension or the cell suspension supernatant is the harvest. Thepurification applied comprises a 4-step purification. Cation exchangechromatography (SP Sepharose Fast Flow (FF)) was used for the capturingstep of G-CSF from the cell culture supernatant, followed by a zincbased immobilized metal affinity chromatography (IMAC) step (Zn-IDAchelating Sepharose Fast Flow (FF)), a second cation exchangechromatography step (Resource S) for polishing and a size exclusionchromatography step (Superdex75) as final step.

Preparation of G-CSF Containing Cell Culture Supernatant

Prior to the capturing step the G-CSF concentration of the supernatantbatches [mg/L] was determined by a G-CSF specific ELISA in order toverify the total G-CSF amount [mg]. The frozen supernatant (−80° C.) wasthawed in a water bath adjusted to 20±5° C. Afterwards the supernatantwas centrifuged at 9000×g for 15 minutes at 4° C. and then additionallyfiltered using 0.2 μg filter units. The pH of the filtered supernatantwas adjusted to pH 4.0 using acetic acid.

Capturing Step (SP Sepharose FF)

An XK 16/20 column was packed with 10 ml of SP Sepharose FF material (1column volume (CV)=10 ml). The SP Sepharose FF resin was obtained fromGE Healthcare (Cat No. 17-0729-01).

The equilibration was performed with 3 CV of the equilibration buffer(20 mM sodium acetate, 100 mM sodium chloride, 0.02% Tween20, pH4.0)followed by loading of the starting material with a flow rate of 2.5ml/min. The following wash step was performed with the same buffer andflow rate, using 5 CV.

The elution was performed with an elution buffer containing 20 mM sodiumacetate, 1 M sodium chloride, 0.02% Tween20, pH4.0, applying a lineargradient from 0% to 40% elution buffer within 8 CV at a flow rate of 2.5ml/min, followed by a step elution with 100% elution buffer with 5 CV.

The G-CSF concentration of the eluate pool, collected from the lineargradient elution, was analysed by a G-CSF specific ELISA.

IMAC Step (Zn-IDA Chelating Sepharose FF)

An XK 16/20 column is packed with 10 ml of Chelating Sepharose FF whichis charged by 2 ml 0.2 M ZnCl₂ (1 column volume (CV)=10 ml). TheChelating Sepharose FF resin was obtained from GE Healthcare (Cat No.17-0575-01). Prior to loading the pH of the IMAC column load (SPSepharose FF eluate) was adjusted to pH 8.0 by NaOH).

The equilibration was performed with 3 CV of the equilibration buffer(20 mM Tris/HCl, 150 mM NaCl, pH 8.0) followed by loading of the SPSepharose FF eluate with a flow rate of 2 ml/min. The following washstep was performed with the same buffer and flow rate, using 2 CV.

The elution was performed with an elution buffer containing 20 mMTris/HCl, 150 mM NaCl, pH 4.0, applying a linear gradient from 0% to100% elution buffer within 3 CV at a flow rate of 1 ml/min. A gradientdelay with 100% elution buffer was applied afterwards with 4 CV.

The G-CSF concentration of the eluate pool, collected from the elutionwith 100% elution buffer, was analysed by a G-CSF specific ELISA.

Polishing Step (Resource S)

A pre-packed ResourceS column (CV=6 ml) from GE Healthcare (Cat No.17-1180-01) column is equilibrated with 5 CV equilibration buffer (20 mMsodium acetate, 0.02% Tween-20, pH 4.0) at a flow rate of 4 ml/min.Prior to purification the IMAC eluate must be adjusted pH to 4.0 withacetic acid and diluted 5-times using the equilibration buffer.

The wash step was performed with 10 CV of the equilibration buffer at aflow rate of 4 ml/min.

The elution was performed with an elution buffer containing 20 mM sodiumacetate, 1 M NaCl, 0.02% Tween-20, pH 4.0, applying a linear gradientfrom 0% to 100% elution buffer within 20 CV at a flow rate of 2 ml/min.

The G-CSF concentration of the eluate pool, collected from the lineargradient elution with 50-85% elution buffer, was analysed by a G-CSFspecific ELISA.

Size Exclusion Chromatography Step (Superdex75)

For the size exclusion step a pre-packed Hiload 26/60 Superdex 75 PrepGrade column was used (GE Healthcare Cat No. 17-1044-01, CV=320 ml). Thecolumn was equilibrated with 1 CV buffer (20 mM sodium acetate, 200 mMNaCl, 0.02% Tween20, pH 6.5) followed by loading of the ResourceS eluateat a flow rate of 2.5 ml/min and a maximum loading volume of 4% CV.

The G-CSF concentration of the eluate pool was analysed by a G-CSFspecific ELISA and by Reversed Phase (RP)-HPLC. The purity of the finalpurification fraction is analysed by use of RP-HPLC, Size Exclusion(SE)-HPLC and SDS-PAGE and is typically >95%.

The size exclusion eluate has been used in some of below described G-CSFexamples.

Purification of rhG-CSF Using Capto MMC Resin as Capture Step Example 1Experiment 1 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.5M NaCl, 0.02% Tween 20,pH 4.0.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) of Capto MMCwas 3 ml.

Buffers

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.1M NaCl, 0.5M arginin monohydrochloride, 0.02% Polysorbate 80, pH 7.0.

Experimental Setup

The column was equilibrated with equilibration buffer followed byloading the start material at a flow rate of 1 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. As seen in Table 1 no G-CSF was found in theflow through fraction. The eluting buffer contained 0.5M arginine monohydrochloride and the pH was altered to 7.0. The recovery of G-CSF was89% and the elution profile was a more concentrated peak. The column wasafter the elution cleaned in place with a 1M NaOH solution. A very smallpeak was visible from the 1M NaOH wash of the column. The chromatogramis showed in FIG. 1.

TABLE 1 Total rhG- Volume rhG-CSF CSF Yield Sample ml μg/ml μg % Startmaterial 8.71 111 966.8 100 Flow through 18 0 0 0 and equil. wash Eluate12 72 864 89

Conclusion

All loaded G-CSF bound to Capto MMC at pH 4.0. A high (89%) yield wasobtained in the elution fraction that was collected in three columnvolumes. The elution buffer had a pH of 7.0 and 0.5M arginine monohydrochloride was included.

Figure legend FIG. 1.

Experiment 1; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. A double peak was obtained by the elution.The double peak was collected as one fraction.

Example 2 Experiment 2 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.5M NaCl, 0.02% Tween 20,pH 4.0.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) of Capto MMCwas 3 ml.

Buffers

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate 80,pH 7.0.

Experimental Setup

The column was equilibrated with equilibration buffer followed byloading the start material at a flow rate of 1 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. As seen in Table 2 no rhG-CSF was found in theflow through. The eluting buffer had a pH of 7.0. More than 90% of therhG-CSF loaded onto the Capto MMC column was found in the elutedfraction. The elution peak was broader than in experiment 1, wherearginin was included in the elution buffer. The elution volume wasdoubled.

TABLE 2 Total rhG- Volume rhG-CSF CSF Yield Sample ml μg/ml μg % Startmaterial 8.92 77.47 691 100 Flow through 30 0 0 0 and equil. wash Eluate24 26.07 626 90.5

Conclusion

Recombinant human G-CSF (rhG-CSF) binds to the Capto MMC resin at pH 4and could be eluted at pH 7 in a solution buffered with sodium citrate.

Example 3 Experiment 3 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.5M NaCl, 0.02% Tween 20,pH 4.0.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) of Capto MMCwas 3 ml.

Buffers

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM HEPES, 0.3M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experimental Setup

The column was equilibrated with equilibration buffer followed byloading the start material at a flow rate of 1 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. As seen in Table 3 no rhG-CSF was found in theflow through. The eluting buffer had a pH of 7.0 and the NaClconcentration was elevated to 0.3M compared to in experiment 2. AllrhG-CSF loaded onto the Capto MMC column was found in the elutedfraction. The chromatography profile is shown in FIG. 2. A broad elutionpeak was obtained, and the whole elution peak was collected as oneelution fraction.

TABLE 3 Total rhG- rhG-CSF CSF Sample Volume ml μg/ml μg Yield % Startmaterial 8.8 71.2 627 100 Flow through 30 0 0 0 and equil. wash Eluate24 27.1 650 103.7

Conclusion

rhG-CSF binds to Capto MMC resin at pH 7 and was eluted to 100% at pH 7when 0.3M NaCl was included in the elution buffer. The elution volumewas large, twice the volume compared to if arginin was included in theelution buffer. This means that 0.3M NaCl does not have the same elutingeffect on G-CSF from a Capto MMC resin as 0.5M arginine.

Figure Legend FIG. 2.

Experiment 3; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.3M NaCl. Adouble peak was obtained by the elution. The broad double peak wascollected as one fraction.

Example 4 Experiment 4 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.5M NaCl, 0.02% Tween 20,pH 4.0.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) was 3 ml.

Buffers

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.02% Polysorbate 80, pH 4.0.

Experimental Setup

The column was equilibrated with equilibration buffer followed byloading the start material at a flow rate of 1 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. As seen in Table 4 no rhG-CSF was found in theflow through. The eluting buffer was altered to a buffer containingarginine and no sodium chloride and the pH was 4.0. No rhG-CSF was foundin the eluted fraction.

TABLE 4 Total rhG- Volume rhG-CSF CSF Yield Sample ml μg/ml μg % Startmaterial 9 69.4 625 100 Flow through 27 0 0 0 and equil. wash Eluate 270 0 0

Conclusion

rhG-CSF could not be eluted from the Capto MMC resin only by adding 0.5Marginine mono hydrochloride to the buffer, without changing the pH. Thiscan work as a wash step.

Example 5 Experiment 5 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.5M NaCl, 0.02% Tween 20,pH 4.0.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) of Capto MMCwas 3 ml.

Buffers

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.1M NaCl, 1M arginin monohydrochloride, 0.02% Polysorbate 80, pH 4.0.

Experimental Setup

The column was equilibrated with equilibration buffer followed byloading the start material at a flow rate of 1 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. The analysis showed that rhG-CSF bound to theCapto MMC resin during theses buffer conditions. As seen in Table 5 norhG-CSF was found in the flow through. The eluting buffer was altered toa buffer containing arginine and no sodium chloride and the pH was 4.0,the same as in the equilibration buffer. No rhG-CSF was found in theeluted fraction. This means a higher arginin concentration (1M) did nothave any effect on the elution of rhG-CSF from the Capto MMC column. Butas seen in FIG. 3 a peak was obtained when the column was eluted by theeluting buffer containing 1M arginin and pH 4. The second large peak isa result of 1M NaOH wash.

TABLE 5 Total rhG- Volume rhG-CSF CSF Yield Sample ml μg/ml μg % Startmaterial 9.04 79.98 723 100 Flow through 18 0 0 0 and equil. wash Eluate9 0 0 0

Conclusion

rhG-CSF could not be eluted from the Capto MMC resin only by adding 1Marginine mono hydrochloride to the buffer, without changing the pH. Thiscan work as a wash step.

Figure legend FIG. 3.

Experiment 5; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium acetate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 4.0 containing 1M argininemono hydrochloride. A double peak was obtained by the elution. A singlepeak was obtained in the eluate fraction. A large peak was obtained bythe 1M NaOH wash.

Example 6 Experiments 6, 7, 8, 9, 10, 11 Start Material

Purified rhG-CSF was diluted in an equilibration buffer to lower thetotal protein concentration and to achieve a more convenient volumeprior to be loaded on a Capto MMC column. And also to achieve the pHthat was requested for each experiment. The rhG-CSF was prior todilution dissolved in 20 mM sodium acetate, 0.2M NaCl, 0.02% Tween 20,pH 6.5.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. A Tricorn 5/150 column (GE Healthcare) was packed with CaptoMMC resin to a bed height of 15 cm. The column volume (CV) of Capto MMCwas 3 ml.

Buffers Experiment 6

Equilibration buffer: 20 mM sodium acetate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Wash buffer: 20 mM NaAc, 1M NaCl, 0.02% Polysorbate 80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experiment 7

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 5.0

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experiment 8

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 6.0

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experiment 9

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 6.5

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experiment 10

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 5.5

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experiment 11

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 4.0

Eluting buffer: 20 mM sodium citrate, 0.5M arginine mono hydrochloride,0.1M NaCl, 0.02% Polysorbate 80, pH 7.0.

Experimental Setup

Each experiment was performed as follows. The purified rhG-CSF wasdiluted approximately 1 in 20 with the equilibration buffer to be used.The pH of the start material was controlled and was in all experimentsthe same as the equilibration buffer without being adjusted. The columnwas equilibrated with equilibration buffer followed by loading the startmaterial at a flow rate of 1 ml/min. This was followed by a wash stepwith the equilibration buffer and then the column was eluted using theeluting buffer. In experiment 6 the column was also washed with a buffercontaining 1M NaCl prior to elution. Samples were withdrawn and analysedfor rhG-CSF by an HPLC method.

As seen FIG. 4 in Experiment 6 no peak was obtained in the 1M NaCl wash,which indicate that no rhG-CSF was washed out from the Capto MMC column.The same elution profile was obtained when G-CSF was purified on a CaptoMMC column independent if a sodium acetate buffer or sodium citratebuffer at pH 4 was used

Experiment 6 and Experiment 11

The results of the analysis to show at which level rhG-CSF binds toCapto MMC and which yield that is obtained is presented in table 6.

TABLE 6 G-CSF in the G-CSF in the Exp. No. pH on the Flow through EluateExperiment load sample % % Figure 6 4.0 0 105 4 11 4.0 0 100 5 7 5.0 098 6 10 5.5 0 97 7 8 6.0 0 97 8 9 6.5 26.8 25.7 9

The data from table 6 shows that G-CSF binds to the Capto MMC resin atpH 4 to 6 without any loss of material in the flow through. But with pH6.5 on the start material, a majority of the G-CSF detected, was foundin the flow through fraction. The Capto MMC column was eluted with abuffer set at pH 7 and containing 0.5M arginine. The G-CSF recovery washigh when the material was loaded to the column at pH 4 to 6, while whenpH 6.5 was used on the start material the yield in the eluate was low.

In the chromatograms below (FIGS. 4, 5 and 6) it is shown that whenrhG-CSF is in a buffer with pH 4 or 5 it is eluted from a Capto MMCcolumn as a double peak. While a single peak was obtained when the pH ofthe start material was 5.5, 6.0 or 6.5 (FIGS. 7, 8 and 9). As shown onthe silver stained SDS PAGE (FIG. 10A) the eluate from Experiment 8 (pH6.0), Experiment 9 (pH 6.5) and Experiment 10 (pH 5.5) have a nicesingle band. While the eluate from Experiment 6 (pH 4.0) and Experiment7 (pH 5.0) shows more bands on the silver stained SDS-PAGE (FIG. 10B).

Conclusion

The results from these experiments shows that rhG-CSF can bind to CaptoMMC at pH 4.0, 5.0, 5.5, 6.0. The rhG-CSF binding to Capto MMC at pH 6.5is less strong and rhG-CSF was found in the flow through fraction whichwas not the case at pH 4 to 6.

The elution profile is better when pH 5.5 to 6.5 was used and theproduct looks better on an SDS-PAGE when these pH was used on theloading material.

Due to the leakage of G-CSF on the Capto MMC column when pH 6.5 wasused, pH 5.5 to 6.0 on the loading material is preferably used.

The bound material was eluted at pH 7 in all experiments, and the peakgot more concentrated when 0.5M arginine mono hydrochloride was includedin the eluting buffer. A 1M NaCl wash can be performed at pH 4 withoutany loss of G-CSF.

Figure legends for FIG. 4-10

FIG. 4.

Experiment 6; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 of the start material using a sodium acetate buffer.The absorbance at 280 nm (mAU) and the conductivity (mS/cm) measured arepresented in the figure. The column was eluted using a buffer with pH7.0 containing 0.5M arginine mono hydrochloride. A double peak wasobtained by the elution. The double peak was collected as one fraction.

FIG. 5.

Experiment 11; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 4.0 using a sodium citrate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. A double peak was obtained by the elution.The double peak was collected as one fraction.

FIG. 6.

Experiment 7; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 5.0 using a sodium citrate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. A double peak was obtained by the elution.The double peak was collected as one fraction.

FIG. 7.

Experiment 10; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 5.5 using a sodium citrate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. One slightly non-uniformed peak wasobtained by the elution.

FIG. 8.

Experiment 8; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 6.0 using a sodium citrate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. One concentrated uniform peak was obtained.

FIG. 9.

Experiment 9; A chromatogram of the G-CSF purification on a Capto MMCcolumn at pH 6.5 using a sodium citrate buffer. The absorbance at 280 nm(mAU) and the conductivity (mS/cm) measured are presented in the figure.The column was eluted using a buffer with pH 7.0 containing 0.5Marginine mono hydrochloride. One concentrated uniform peak was obtained.

FIG. 10A and FIG. 10B

Experiment 6, 7, 8, 9, 10; Separation of proteins in the start andeluate from the Capto MMC experiments where different pH values was usedin the start material. The samples are reduced (SDS treated) andseparated on a 10% polyacryl amide gel. The proteins are the visualizedby a silver staining.

Example 7 Experiment 12 Start Material

Recombinant human G-CSF produced in HEK 293 cells. The cells wereremoved and the cell free supernatant was the start material loaded ontothe Capto MMC column.

Chromatographic Resin and Column

Capto MMC, a mixed mode resin from GE Healthcare (cat no. 17-5317), wasused as capture step for the rhG-CSF molecule. Capto MMC is a weakcationic resin with hydrophobic and thiophilic interactions and hydrogenbonding. An XK16 column (GE Healthcare) was packed with Capto MMC resinto a bed height of 13.5 cm. The column volume (CV) of Capto MMC was 27ml.

Buffers

Equilibration buffer: 20 mM sodium citrate, 0.1M NaCl, 0.02% Polysorbate80, pH 6.0

Eluting buffer: 20 mM sodium citrate, 0.1M NaCl, 0.5M arginin monohydrochloride, 0.02% Polysorbate 80, pH 7.0.

Experimental Setup

The pH of the cell free supernatant was adjusted to 6.0. The column wasequilibrated with equilibration buffer followed by loading the pHadjusted start material at a flow rate of 13.5 ml/min. This was followedby a wash step with the equilibration buffer and then the column waseluted using the eluting buffer. Samples were withdrawn and analysed forrhG-CSF by a HPLC method. The analysis showed that all rhG-CSF loadedonto the column bound to the Capto MMC resin during theses bufferconditions, no G-CSF was found in the flow through fraction. The elutingbuffer contained 20 mM NaCitrate, 0.5M arginine, 0.1M NaCl and 0.02%Polysorbate 80, and the pH was 7.0. The elution of the bound proteins tothe Capto MMC resin resulted in a main peak and a small second peak. AllG-CSF loaded onto the Capto MMC column was found in the main peak and noG-CSF was detected in the second small peak (FIG. 11).

Conclusion

Recombinant human G-CSF in cell free cultured medium bound to a CaptoMMC resin at pH 6.0.

No G-CSF was found in the flow through fraction.

The bound material was eluted from the Capto MMC resin by altering thepH to 7 and by adding 0.5M arginine to concentrate the elution peak.

Figure legend FIG. 11

Experiment 12; A chromatogram of the G-CSF purification from a cell freesupernatant on a Capto MMC column at pH 6.0 using a sodium citratebuffer. The absorbance at 280 nm (mAU) and the conductivity (mS/cm)measured are presented in the figure. The column was eluted using abuffer with pH 7.0 containing 0.5M arginine mono hydrochloride. A doublepeak was obtained but G-CSF was only found in the main peak.

Example 8 Column and Resin

A Tricorn 5/50 column (GE Healthcare) was packed with a yeast derivedFab fragment based affinity ligand coupled to a Capto MP base matrix.The bed height was approximately 2 cm, giving a resin volume of app. 0.4ml. The affinity resin prototype (G-CSF8) was obtained from BAC BV.

Starting Material

The starting material used was a G-CSF containing cell supernatantproduced in HEK293F cells.

Buffer Compositions: Buffer A (Equilibration Buffer)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 6.0, conductivity 32mS/cm at +25° C.

Buffer B (Elution Buffer I)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 3.0, conductivity 32mS/cm at +25° C.

The column was equilibrated with equilibration buffer A followed byloading of the starting material. The resin was thereafter washed withequilibration buffer A and the bound G-CSF was subsequently eluted withelution buffer B. The G-CSF content in the starting material and eluatewere analyzed (Table 7).

TABLE 7 Results from G-CSF affinity resin experiment, elution with pH 3Total Volume G-CSF amount G- Yield Sample (ml) (ug/ml) CSF (ug) (%)Starting material 100 2 200 100 (load) Eluate (Buffer B) 1.5 111 170 83

FIG. 12 shows a Coomassie stained SDS-PAGE showing the eluate after theaffinity chromatography step.

Lane Sample

1 Molecular weight standard

2 Eluate (Example 8)

Conclusion Example 8

Excellent purity and recovery was achieved when using a low pH buffer(pH 3) for elution of G-CSF.

Example 9 Column and Resin

A Tricorn 5/50 column (GE Healthcare) was packed with a yeast derivedFab fragment based affinity ligand coupled to a Capto MP base matrix.The bed height was approximately 2 cm, giving a resin volume of app. 0.4ml. The affinity resin prototype (G-CSF8) was obtained from BAC BV.

Starting Material

The starting material used was a G-CSF containing cell supernatant fromHEK293F cells.

Buffer Compositions: Buffer a (Equilibration Buffer)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 6.0, conductivity 32mS/cm at +25° C.

Buffer C (Elution Buffer II)

1.0M NaCl, 0.02M Na-citrat, 0.8M Arg, 0.02% Tween 20, pH 6.0,conductivity 89 mS/cm at +25° C.

The equilibration and elution buffers are not limited to the stated pH,concentrations, and type of buffer, salts or detergent.

The column was equilibrated with equilibration buffer A followed byloading of the starting material. The resin was thereafter washed withequilibration buffer A and the bound G-CSF was subsequently eluted withelution buffer C. The G-CSF content in the starting material and eluatewere analyzed (Table 8).

TABLE 8 Results from G-CSF affinity resin experiment, elution witharginine Total Volume G-CSF amount G- Yield Sample (ml) (ug/ml) CSF (ug)(%) Starting material 100 2 200 100 (load) Eluate (Buffer B) 3.7 30 11056Conclusion example 9

It is possible to elute G-CSF from the affinity column using a mixtureof sodium chloride and arginine.

Example 10 Column and Resin

A Tricorn 5/50 column (GE Healthcare) was packed with a yeast derivedFab fragment based affinity ligand coupled to a Capto MP base matrix.The bed height was approximately 2 cm, giving a resin volume of app. 0.4ml. The affinity resin prototype (G-CSF8) was obtained from BAC BV.

Starting Material

The starting material used was a G-CSF containing cell supernatant fromHEK293F cells.

Buffer Compositions: Buffer a (Equilibration Buffer)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 6.0, conductivity 32mS/cm at +25° C.

Buffer D (Elution Buffer III)

2.0M MgCl2, 0.02M Tris, 0.02% Tween 20, pH 7.5, conductivity 144 mS/cmat +25° C.

The equilibration and elution buffers are not limited to the stated pH,concentrations, and type of buffer, salts or detergent.

The column was equilibrated with equilibration buffer A followed byloading of the starting material. The resin was thereafter washed withequilibration buffer A and the bound G-CSF was subsequently eluted withelution buffer D. The G-CSF content in the starting material and eluatewere analyzed (Table 9).

TABLE 9 Results from G-CSF affinity resin experiment, elution with MgCl2Total Volume G-CSF amount G- Yield Sample (ml) (ug/ml) CSF (ug) (%)Starting material 100 12 1200 100 (load) Eluate (Buffer B) 2.5 390 97081

Conclusion Example 10

It is possible to elute G-CSF from the affinity column using 2 M MgCl2as eluent.

Example 11 Column and Resin

Tricorn 5/50 columns (GE Healthcare) was packed with 3 different yeastderived Fab fragment based affinity ligand prototypes, coupled to aCapto MP base matrix. The bed height was approximately 2 cm, giving aresin volume of app. 0.4 ml. The affinity resin prototypes (G-CSF2,G-CSF3 and G-CSF6) were obtained from BAC BV.

Starting Material

The starting material used was a G-CSF containing cell supernatant fromHEK293F cells.

Buffer Compositions: Buffer A (Equilibration Buffer)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 6.0, conductivity 32mS/cm at +25° C.

Buffer B (Elution Buffer I)

0.3M NaCl, 0.02M Na-citrat, 0.02% Tween 20, pH 3.0, conductivity 32mS/cm at +25° C.

The columns were equilibrated with equilibration buffer A followed byloading of the starting material. The resins were thereafter washed withequilibration buffer A and the bound G-CSF was subsequently eluted withelution buffer B. The G-CSF content in the starting material, flowthrough fractions and eluates were analyzed (Table).

TABLE 10 Results from G-CSF affinity resin experiment, test of differentaffinity ligand prototypes, elution with pH 3 Total Volume G-CSF amountG- Yield Sample (ml) (ug/ml) CSF (ug) (%) Ligand G-CSF2 Startingmaterial 100 10 1100 100 (load) Flow through fraction 112.5 4 500 45Eluate (Buffer B) 2 290 570 57 Ligand G-CSF3 Starting material 100 111100 100 (load) Flow through fraction 112.5 0 0 0 Eluate (Buffer B) 2510 1020 93 Ligand G-CSF6 Starting material 100 12 1200 100 (load) Flowthrough fraction 112.5 2 200 19 Eluate (Buffer B) 1.5 450 670 56

FIG. 13 shows a silver stained SDS-PAGE showing starting material, flowthrough and eluates after the affinity chromatography step.

Legend: Lane Sample

1 G-CSF2 start; 2 G-CSF2 flow through; 3 Blank; 4 G-CSF2 eluate; 5.Blank; 6 G-CSF3 flow through; 7 Blank; 8 G-CSF3 eluate; 9. Blank; 10G-CSF6 flow through; 11 Blank 12 G-CSF6 eluate

Conclusion Example 11

Different affinity ligand prototypes gives different binding capacity ofG-CSF, as shown by the detection of G-CSF in the flow through and thevarying recovery in the elution fraction using low pH as eluent.However, as shown in FIG. 13, all affinity ligand gives the sameexcellent purity profile in the eluate.

1. A process of purifying a Growth Factor Protein Granulocyte ColonyStimulating Factor (G-CSF) in a purification sequence employingchromatography comprising: performing at least one chromatogrpahy stepusing a multimodal resin which comprises a negatively charged2-(benzoylamino) butanoic acid ligand, binding the G-CSF to themultimodal resin at a pH from 4 to 6.2, and eluting the G-CSF at a pH inthe range between 5.5 to 6.5, wherein the elution is performed with anarginine buffer having a concentration in the range of from 0.1 M to 2.0M, optionally in combination with an affinity ligand chromatography stepwherein the affinity ligand is a yeast derived F_(ab) fragment directedtoward the G-CSF.
 2. The process of claim 1, wherein the multimodalresin comprises moieties bound to a matrix and the moieties are able tointeract with the G-CSF in a mixture by ionic interactions, hydrogenbonding, and/or hydrophobic interaction.
 3. The process of claim 2,wherein the mixture comprising the G-CSF is a solution.
 4. The processof claim 1, wherein the G-CSF is a recombinant G-CSF.
 5. The process ofclaim 1, wherein the G-CSF binds to the multimodal resin at pH 4.0 to pH6.0, and the G-CSF is eluted from the multimodal resin at pH 6.5.
 6. Theprocess of claim 1, wherein the binding and/or elution steps are carriedout in a buffering substance comprising sodium citrate, sodium acetate,or 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid (HEPES). 7.The process of claim 6, wherein the non-ionic detergent is present inany of the buffers.
 8. The process of claim 1, wherein a buffercomprising about 0.1 M to about 2 M sodium chloride or about 0.1 M toabout 2 M arginine mono hydrochloride is used in a wash-step at a pHrange of 4.0 to 6.0 prior to elution of the G-CSF.
 9. The process ofclaim 1, further comprising applying a wash buffer to the multimodalresin, to wash away contaminants and retain the G-CSF, before the G-CSFis eluted, wherein the wash buffer comprises an amino acid having abasic side chain and/or a salt.
 10. The process of claim 1, wherein thepurification sequence further comprises one or more of the followingsteps: subjecting the G-CSF to a chemically based inactivation step forenveloped viruses; binding and elution from an affinity resin comprisingas an affinity ligand a yeast derived F_(ab) fragment directed towardthe G-CSF; binding and elution from a cation exchanger; subjecting theG-CSF to a pathogen filtration removal step with a filter having a meanpore size of about 20 nm; subjecting the G-CSF to a buffer exchangeand/or concentrating step comprising ultra-filtration with anapproximate cut off value of between 1 and 5 kDa; and subjecting theG-CSF to a size exclusion chromatography resin.
 11. The process claim10, wherein the purity of the product resulting from the affinitychromatography step is more than 90%.
 12. The process claim 11, whereinadditional chromatography step(s) is/are performed, selected from sizeexclusion, anion exchange, cation exchange, hydrophobic interaction andimmobilized metal affinity chromatography, wherein the purity of thefinal product is more than 99%.
 13. The process of claim 1, wherein thepurification sequence further comprises pathogen removal and/orinactivation steps.
 14. The process of claim 1, comprising subjectingthe G-CSF to a chromatography step using as an affinity ligand a yeastderived F_(ab) fragment directed toward the G-CSF.
 15. The process ofclaim 1, wherein the binding step is carried out in a buffer comprisingat least one of the buffering substances selected from the groupconsisting of sodium citrate, histidine,2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid (HEPES),2-(N-Morpholino)ethane sulfonic acid (MES), Tris base and sodiumacetate.
 16. The process of claim 7, wherein the non-ionic detergent isselected form the group consisting of Polysorbate 20, Polysorbate 40,Polysorbate 60, and Polysorbate
 80. 17. The process of claim 1, whereinthe arginine buffer further comprises at least one of the bufferingsubstances selected from the group consisting of sodium citrate,histidine, 2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethane sulfonic acid(HEPES), 2-(N-Morpholino)ethane sulfonic acid (MES), Tris base andsodium acetate.